Compartmentation and cellular environment

The 31P-NMR spectra of fungal and plant tissues generally contain a number of identifiable resonances: three arising from the a, ¡5, and y phosphates of ATP (and other NTPs); two Pi resonances arising from the cytosolic and vacuolar phosphates with a pH-dependent chemical shift; and sugar phosphoester peaks. In addition to Pi, the spectra of ectomycorrhizal fungi show resonances from the terminal and penultimate phosphates of PolyP (Fig. 3).

One of the earliest and still most widespread uses of 31P-NMR has been the measurement of intracellular pH (Roberts et al., 1980). Several methods are available to measure the intracellular pH in living cells— micro-pH electrodes, fluorescent dyes, the distribution of weak acids or bases and NMR are the most commonly used techniques. The first three pp

Fig. 3. Representative 31P-NMR spectrum at 162 MHz of C. geophilum mycelium (approx. 0.5 g fresh weight). 4000 scans were accumulated with a recycle time of 2 s and a 90 0 pulse. The peak assignments are denoted: Pic, cytoplasmic inorganic phosphate; Piv, vacuolar inorganic phosphate, PP, inner phosphates of polyphosphates.

Fig. 3. Representative 31P-NMR spectrum at 162 MHz of C. geophilum mycelium (approx. 0.5 g fresh weight). 4000 scans were accumulated with a recycle time of 2 s and a 90 0 pulse. The peak assignments are denoted: Pic, cytoplasmic inorganic phosphate; Piv, vacuolar inorganic phosphate, PP, inner phosphates of polyphosphates.

methods are all invasive and have the potential of bringing about undesired alterations inside the cell. NMR is the only method for determining the intracellular pH which can be completely non-invasive. For this purpose, any NMR-detectable nucleus which is in close proximity to a protonation state in an intracellular weak acid can generally be utilized as an NMR probe. Inorganic phosphate is the most popular probe to determine intracellular pH since it is well resolved, easily identified, and usually occurring in large concentrations. At neutral pH, inorganic phosphate exists mainly in the H2P04~ form (at 0.58 ppm) and the divalent form HP042_ (at 3.14 ppm).

If the pH difference between any of the cell compartments (cytosol, mitochondria, vacuoles, etc.) is larger than 0.3 pH units, Pi resonances will be resolved. For example, there are two Pi peaks in the in vivo spectrum of C. geophilum (Fig. 3), whereas only one resonance is observed in the spectrum of the fungal extract (not shown). The lower-field Pi resonance was assigned to Pi originating from the cytosolic pH. Pi in organelles such as mitochondria is usually neglected since it appears to be present in an NMR-invisible form. Roberts and Jardetzky (1981) have shown that the pATa of Pi is sensitive to the ionic strength of the solution. However, Gillies et al. (1981) have pointed out that this dependence, within the physiological range, is very small, corresponding to less than 0.1 pH unit. Several pH-dependent phosphomonoesters can function as alternative pH probes including cellular metabolites (fruc-tose-l-phosphate, glucose-6-phosphate, polyphosphates, etc.) and chemicals (methylenediphosphonic acid, etc.). Tissue pH has now been measured in many non-mycorrhizal roots. The cytoplasmic pH of corn and pea root tips was 7.0-7.4 while the vacuolar pH was 5.5-6.0 (Loughman and Ratcliffe, 1984). In ectomycorrhizal fungi the cytoplasmic pH was 6.0-6.5 whereas the vacuolar pH was acidic (5.0) (F. Martin, unpubl. data). The importance of maintaining the tissue in a well aerated medium in the NMR tube has been stressed (Loughman and Ratcliffe, 1984).

The large size of the vacuole in mature roots and fungal cells prevents the analysis of cytoplasmic metabolites. In spectra of ectomycorrhiza and ectomycorrhizal mycelium (Fig. 3), Pi and PolyP resonances were the only significant resonances (Martin et al., 1983, 1985b; Loughman and Ratcliffe, 1984; Grellier et al., 1989). Monitoring of cytoplasmic events by NMR therefore requires samples containing an important cytosolic compartment. It is for this reason that many of the high-resolution NMR investigations on roots have been carried out using root tips (see Loughman and Ratcliffe, 1984).

Excess intracellular phosphate is stored as PolyP in most fungi, including those that are ectomycorrhizal (Martin et al., 1983, 1985b). This large amount of PolyP gives rise to prominent resonances in the 31P-NMR spectrum of these micro-organisms and NMR is thus an excellent approach for investigating PolyP metabolism and PolyP physicochemical properties in mycorrhizal tissue. Most PolyP (80%) occurs in mycorrhizal fungi as oligophosphates with an average chain length of 10 phosphate residues (Martin et al., 1983). 31P-NMR has been used to estimate the quantities of PolyP in the ectomycorrhizal fungi C. geophilum, Hebeloma crustuliniforme (Martin et al., 1983, 1985b), Paxillus involutus (Grellier et al., 1989) and Pisolithus tinctorius (Tillard et al., 1990). The in vivo physicochemical state of PolyP was determined by Martin et al. (1985b). Comparison of measured nuclear magnetic relaxation parameters, such as resonance linewidths and spinlattice relaxation times, Tx, of PolyP in vivo, with those observed in model solutions, suggested that PolyP exist in mycorrhizal fungi as aggregates with reduced correlation time.

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